Toxicological, physiological, and biochemical information gained from tissue culture studies must be interpreted with caution since cells in culture are different from the parent cell type found in vivo. The loss of specific differentiated functions and the presence of abnormal karyotypes, biochemical characteristics, and hormonal responses are common features of established cell lines. Although some of these limitations may be overcome by using cells in primary culture, primary cultures quickly switch from aerobic to glycolytic metabolism and eventually dedifferentiate. The loss of aerobic metabolism and differentiated functions alters the relationships and interdependence of cellular processes which limits extrapolations between in vitro and in vivo studies. Since tho dedifferentiation that occurs during primary culture may be a consequence of changes in cellular energy metabolism and the extracellular environment, the first goal of this proposal is to examine the role of aerobic metabolism and the extracellular environment in maintaining differentiated function. Using primary cultures of renal proximal tubule cells, standard culture conditions will be modified to maintain aerobic metabolism (Specific Aim 1) and to more closely reproduce the extracellular environment of the proximal tubule cell in vivo (Specific Aim 2). The model systems used for these studies will be proximal tubule cells grown on Millicell culture inserts (SHAKE and MICROPERFUSED). Using diverse markers such as morphology, hormonal responsiveness, and functionally specific enzyme markers and transporter systems, the degree of differentiated function maintained in these systems will be compared against freshly isolated proximal tubules and primary cultured proximal tubule cells grown using standard culture conditions (highest vs. lowest metabolic and differentiated state, respectively). The second goal of this proposal is to investigate the mechanism of aryl-glutathione conjugate-induced nephrotoxicity in the newly developed culture system which maintains metabolic and differentiated functions similar to the proximal tubule cell in vivo (Specific Aim 3). This proposal will investigate some of the current issues regarding maintenance of differentiated functions in primary culture and develop a superior culture system for more direct and relevant comparisons between toxicology, physiology, and biochemistry studies in vitro and studies in vivo. Additionally, information gained from these studies can be applied to other organ types and animal species to reduce the number of vertebrate animals used in research.